1. Field of the Invention
The present invention relates to the field of standard or reference materials useful in assay procedures, and more particularly relates to materials and methods for the preparation of such standards which do not display significant turbidity.
2. Description of the Prior Art
The use of serum standards or references in blood chemistry analysis is well known. It is frequently advantageous as a diagnostic aid to determine the level of certain constituents of a patient's blood. This determination is made with the aid of serum standards. The serum standards are commonly stored as a dry powder after lyophilization to be reconstituted at the time of use. Alternatively, they may be frozen and thawed. It is desirable that the lyophilized serum, when reconstituted with an aqueous media, or the frozen serum when thawed, be stable and have substantial optical clarity to minimize interference with the analytical measurement of serum constituents.
Two important blood constituents are triglyceride and cholesterol. It is therefore desirable to prepare a serum standard or a reference material containing triglyceride and/or cholesterol and having good optical clarity for use with analytical testing procedures. The serum should also be stable and easily reconstituted after storage.
The difficulties in photometric analysis which result from turbidity in serum and plasma samples are generally set forth in U.S. Pat. No. 3,853,465, issued to Ruch et al. on Dec. 10, 1974, which is hereby incorporated by reference. In this regard, in our U.S. Pat. No. 3,955,925, issued on May 11, 1976, we disclosed a method for preparing a serum standard which remains stable and optically clear upon lyophilization and reconstitution with aqueous media. In accordance with the method disclosed therein, the pre-beta lipoproteins, beta lipoproteins and chylomicrons are (turbidity potential lipoproteins) removed from human serum by specific precipitation with a metal cation and a polysulfate. Further, in our U.S. Pat. No. 4,045,176, issued on Aug. 30, 1977, we disclosed a method for the preparation of a serum standard having normal or elevated levels of cholesterol and/or triglyceride and being stable and optically clear upon lyophilization and reconstitution. In accordance with the method of the latter patent, isolated non-primate lipoproteins or isolated high density human lipoproteins are added to serum, the serum in certain embodiments having the pre-beta lipoproteins, beta lipoproteins and chylomicrons removed therefrom.
In U.S. Pat. No. 3,274,062, issued to Lou on Sept. 20, 1966, there is disclosed a method for concentrating cholesterol-rich protein constituents of human blood serums. In accordance with the Lou method, the beta lipoproteins are precipitated from human plasma or serum by the addition of "Mepesulfate" and calcium ions. The precipitate is then purified by dialysis and subsequently added to liquid serum to form a high cholesterol standard. The Lou method does not involve the use of a lipoprotein solution to which preservatives have been added as a diluent, and moreover does not disclose the addition of this lipoprotein diluent to a lyophilized serum base. In fact, the supernatant serum from which the beta lipoproteins are removed according to the Lou method is discarded as waste material.
A method for isolating triglyceride-rich concentrate and cholesterol-rich precipitate from egg yolks and human plasma, respectively, is disclosed in U.S. Pat. No. 3,764,556, issued to Kuchmak et al. on Oct. 9, 1973. In accordance with the Kuchmak et al. disclosure, a cholesterol-rich lipid precipitate isolated from human plasma and containing about 75% of the total cholesterol and about 60% of the triglyceride was added to bovine, horse and human serums to provide a control standard having the desired level of lipids. As reported therein, the serum preparations were frozen and thawed three times with the solutions retaining their original transparent appearance. The Kuchmak et al. patent indicates that the lipid fractions must be immediately dissolved in serum at room temperature. The Kuchmak et al. reference does not disclose the inclusion of a preservative with a lipoprotein diluent, nor the adding of a lipoprotein diluent to a lyophilized serum base to provide a serum standard which is free of turbidity and has the desired levels of lipoproteins. Further, there is no teaching in the Kuchmak et al. reference that the lipoprotein fraction be used as a diluent for lyophilized, serum to provide a resulting serum standard which does not display turbidity.